The overall goal of this application is to understand further the molecular mechanisms involved in the autoimmune response to beta cell autoantigens and their relevance in the development of type 1 diabetes (T1D). We will focus on understanding the interaction between human autoreactive B and T cells in the development of autoimmunity to a major autoantigen, the enzyme glutamic acid decarboxylase 65 (GAD65). We furthermore propose to generate a novel humanized mouse model of diabetes using GAD65 as the primary target. The in vitro B-T cell interaction studies will be then addressed at the in vivo level for better understanding of the pathways resulting in T1D. Aim 1: Test the hypothesis that GAD65 epitope specificity of autoreactive B cells affects the presentation of antigen to DR and DQ restricted T cells in type 1 diabetes. Antigen presentation to T cells by antigen-specific EVB-transformed B cell lines can be enhanced or suppressed based on the epitope specificity of the surface immunoglobulin on B cells. We have recently shown that GAD65-specific monoclonal antibodies inhibit the presentation of major DRBI*0401 restricted T cell epitopes localized within their binding region. Using this novel approach we will study how epitope specificity of autoreactive B cells to GAD65 affect the presentation to DR4 (DRB1*0401 and 0405), or DQ8 (DQB1*0302) restricted T cell hybridomas. Aim 2: Test the hypothesis that induction of strong Thl and CTL dominated immune responses to GAD65 can result in a cells destruction and diabetes in transgenic mice carrying DR4 and/or DQ8 and over expressing GAD65 at the a cell level. We propose crossing the RIP7-GAD65 mouse line with the DR4/DQ8 expressing line to generate mice with human MHC II diabetes susceptibility genes that also express a human early diabetes autoantigen in human quantities. If spontaneous diabetes ensues when double homozygosity is achieved, we will study mechanisms of disease development. However, for the purpose of this application we speculate we would need to induce disease development by immunizing DR/DQ-RIP7-GAD65 transgenics with GAD65 antigen. The nature of the generated immune response will determine how to proceed if insulitis and diabetes are not the immediate outcome.